U.S. patent application number 10/057753 was filed with the patent office on 2006-08-10 for method for labeling dna and rna.
Invention is credited to Sergei Bavykin, Andrei D. Mirzabekov.
Application Number | 20060178508 10/057753 |
Document ID | / |
Family ID | 25022921 |
Filed Date | 2006-08-10 |
United States Patent
Application |
20060178508 |
Kind Code |
A9 |
Bavykin; Sergei ; et
al. |
August 10, 2006 |
Method for labeling DNA and RNA
Abstract
A method for fragmenting and labeling nucleic acids is provided.
The method comprises maintaining double- and single-stranded
nucleic acid molecules in an aerobic or an anaerobic atmosphere,
contacting the molecules with hydrogen peroxide and radical
generating coordination complexes for a time and at concentrations
sufficient to produce aldehyde moieties on the molecules, reacting
the aldehyde moieties with amine to produce a condensation product,
and labeling the condensation product.
Inventors: |
Bavykin; Sergei; (Darien,
IL) ; Mirzabekov; Andrei D.; (Moscow, RU) |
Correspondence
Address: |
CHERSKOV & FLAYNIK
THE CIVIC OPERA BUILDING
20 NORTH WACKER DRIVE, SUITE 1447
CHICAGO
IL
60606
US
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20020165388 A1 |
November 7, 2002 |
|
|
Family ID: |
25022921 |
Appl. No.: |
10/057753 |
Filed: |
January 23, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09751654 |
Dec 29, 2000 |
6818398 |
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10057753 |
Jan 23, 2002 |
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60263840 |
Jan 23, 2001 |
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Current U.S.
Class: |
536/25.32 |
Current CPC
Class: |
B01J 20/283 20130101;
C12Q 1/6806 20130101; C07H 21/00 20130101; C12Q 1/6816 20130101;
C12Q 1/6806 20130101; C12Q 2563/113 20130101; C12N 15/101 20130101;
C12Q 2563/107 20130101 |
Class at
Publication: |
536/025.32 |
International
Class: |
C07H 21/04 20060101
C07H021/04 |
Goverment Interests
CONTRACTUAL ORIGIN OF THE INVENTION
[0002] The United States Government has rights in this invention
pursuant to Contract Number W-31-109-ENG-38 between the United
States Government and Argonne National Laboratory.
Claims
1. A method for labeling nucleic acids, the method comprising: a)
contacting nucleic acid molecules with hydrogen peroxide and a
redoxactive coordination complex for a time and at concentrations
sufficient to produce free-aldehyde moieties on the molecules; b)
reacting the aldehyde moieties with amine to produce a condensation
product; and c) labeling the condensation product.
2. The method as recited in claim 1 wherein the step of labeling
the condensation product further comprises: a) reducing the
condensation product; and b) contacting the reduced condensation
product with a chromophore.
3. The method as recited in claim 1 wherein the nuclease is a
coordination complex selected from the group consisting of
1,10-phenanthroline-Cull, bleomycin-Fe(III), EDTA-Fe, ascorbic
acid-Cu, methylene-blue-Cu, metallophorphyrin, or combinations
thereof.
4. The method as recited in claim 1 wherein the amine is a primary
amine.
5. The method as recited in claim 1 wherein the amine is ethylene
diamine or hydrazine or aminated biotin.
6. The method as recited in claim 1 wherein the contacting step
occurs in an anaerobic environment.
7. The method as recited in claim 1 wherein the step of labeling
the condensation product further comprises reducing the
condensation product and cross-linking the reduced condensation
product with a label in one reaction step.
8. The method as recited in claim 1 wherein the step of contacting
the nucleic acid molecules with redox-active coordination complex
includes contacting the nucleic acid with a denaturing agent.
9. A method for modifying nucleic acids, the method comprising: a)
contacting free radicals with the nucleic acids to produce free
nucleic acid bases and aldehyde forms of ribose and deoxyribose; b)
contacting the aldehyde forms with an amine to produce a
condensation product; c) reducing the condensation product; and d)
labeling the reduced condensation product.
10. The method as recited in claim 9 wherein the step of producing
free radicals comprises reacting hydrogen peroxide with chemical
nucleases.
11. The method as recited in claim 10 wherein the chemical
nucleases are coordination complexes selected from the group
consisting of 1,10-phenanthro-line-Cull, bleomycin-Fe(III),
EDTA-Fe, ascorbic acid-Cu, methylene-blue-Cu, metallophorphyrin, or
combinations thereof.
12. The method as recited in claim 9 wherein steps d and e occur
simultaneously.
13. The method as recited in claim 9 wherein step e occurs in
anaerobic conditions.
14. The method as recited in claim 9 wherein the nucleic acid is
double stranded and wherein the step of contacting the free
radicals with the nucleic acids is preceded by the addition of a
double-strand weakening agent.
15. The method as recited in claim 14 wherein the double-strand
weakening agent is a denaturing agent selected from the group
consisting of carbonic acid, urea, ethyl carbonate, cyanamide,
urethane, and combinations thereof.
16. The method as recited in claim 9 wherein the nucleic acid is
modified at temperatures below the boiling point of water.
17. The method as recited in claim 9 wherein the nucleic acid
modification occurs at between 0.degree. C. and 95.degree. C.
18. The method as recited in claim 9 wherein the free radicals are
contacted with the nucleic acids in an anaerobic atmosphere.
Description
[0001] This application is based on Provisional Application No.
60/263,840 filed Jan. 23, 2001.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] This invention relates to a method for labeling DNA and RNA,
and more specifically, this invention relates to a method for
labeling DNA and RNA using radical-producing chemical agents.
[0005] 2. Background of the Invention
[0006] DNA microchip technology is a rapid, high throughput
platform for nucleic acid hybridization reactions. However, nucleic
acid fragmentation and labeling are two of the limiting steps in
the development of rapid protocols for DNA microchip
technology.
[0007] PCR and other amplification techniques are utilized for
bacteria identification. Immunological methods and
mass-spectrometry also have been adapted for this purpose, but are
expensive and cumbersome.
[0008] Several enzymatic and chemical protocols are available for
fluorescent labeling of nucleic acids. All of these methods are
expensive and time consuming. Most of these protocols demand
careful prerequisite nucleic acid isolation, fractionation
(generally requiring one or more hours), labeling, separate sample
fragmentation procedures and a final purification step.
[0009] Typical nucleic acid labeling methods adopt a myriad of
approaches. For example, M. D. Schena et al., Science 270, 467-470
(1995); J. L. DeRisi et al., Science 278, 680-686 (1997); G. P.
Yang et al., Nucl. Acid Res. 27, 1517-1523 (1999); K. Wang et al.,
Gene 229, 101-108 (1999), and M. Wilson et al. Proc. Natl. Acad.
Sci USA 96, 12833-12838 all rely on effecting labeling using
reverse transcriptase. Typically, this process requires from one to
two hours to complete.
[0010] D. Guiliano et al. BioTechniques 27 146-152 (1999) and G. T.
Hermanson, Bioconjugate Techniques (Academic Press, Inc. San Diego,
Calif., 1996) utilize random priming. However, these protocols
require from 3 to 10 hours to complete.
[0011] Terminal transferase protocols are featured in K. L.
Gunderson et al. Genome Res. 8, 1142-1153 (1998) and L. Wodicka et
al. Nat. Biotechnol. 15, 1359-1367. However, these processes also
require between 1 and 2 hours to run.
[0012] Polymerase Chain Reaction (PCR) protocols for labeling are
widespread. Typical references for PCR processes include R. J.
Sapolsky et al. Genomics 33, 445-456 (1996); M. T. Cronin et al.
Hum. Mutat. 7, 244-255 (1996); S. Tyagi et al. Nat. Biotechnol 16,
49-53 (1998); and P. N. Gilles et al. Nat. Biotechnol 17, 365-370
(1999). However, PCR protocols require between 1 and 2 hours to
complete.
[0013] A need exists in the art for a simple protocol for labeling
nucleic acids found either in DNA or RNA. The protocol should
require mild conditions of reaction and should yield high amounts
of cross-linked complexes in short incubation times. The method
should facilitate both the labeling and fragmentation at random
sites of nucleic acids, therefore being independent of sequence or
two dimensional structures. The method should facilitate the
end-labeling of nucleic acids and further accommodate a broad
number of label derivatives, the later to be attached to nucleic
acids. Lastly, the method should accommodate automated
processes.
SUMMARY OF THE INVENTION
[0014] It is an object of the present invention to provide a method
for labeling nucleotide molecules that overcomes many of the
disadvantages of the prior art.
[0015] Another object of the present invention is an economical
method for labeling DNA and RNA molecules. A feature of the
invention is that a product of the labeling method is a Schiff base
comprising the nucleic acid and the label. An advantage of the
method is that the method is independent of nucleic acid sequences
of the probe. Another advantage is that the method facilitates
manipulation of both DNA and RNA.
[0016] Yet another object of the present invention is to provide a
method for modifying nucleic acid. A feature of the invention is
that the modification occurs aerobically and anaerobically in the
presence of hydrogen peroxide to ultimately lead to the formation
of a Schiff base for subsequent labeling. An advantage of the
present method is that the Schiff base is reduced and labeled
simultaneously to provide a streamlined nucleic acid and labeling
protocol.
[0017] Briefly, the invention provides a method for labeling
nucleic acids, the method comprising maintaining double-stranded
nucleic acid molecules in an aerobic and anaerobic atmosphere;
contacting the molecules with hydrogen peroxide and nuclease for a
time and at concentrations sufficient to produce aldehyde moieties
on the molecules; reacting the aldehyde moieties with amino
derivatives of fluorophores, or with any other label containing
primary amino groups, or with an amine to produce a condensation
product; and labeling the condensation product.
BRIEF DESCRIPTION OF THE DRAWING
[0018] The present invention together with the above and other
objects and advantages may best be understood from the following
detailed description of the embodiment of the invention illustrated
in the drawing, wherein:
[0019] FIG. 1 is a reaction sequence of DNA labeling, in accordance
with features of the present invention;
[0020] FIG. 2 is a reaction sequence of RNA labeling, in accordance
with features of the present invention;
[0021] FIG. 3 is an illustration of fragmentation and hybridization
of B. thuringiensis, using the invented protocol in accordance with
features of the present invention;
[0022] FIGS. 4A-B are illustrations of the electrophoresis of
labeled/fragmented nucleic acids under denaturing conditions, with
(i) showing fluorescence under direct light and (ii) showing
ethidium bromide stained gel;
[0023] FIG. 5 is a comparison for different fragmentation
protocols, in accordance with features of the present
invention;
[0024] FIGS. 6A-C illustrate the effect of reducing agent on
fragmented and labeled nucleic acids, in accordance with features
of the present invention;
[0025] FIGS. 7A-B illustrate ethidium bromide stained gel images,
in accordance with features of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0026] A method for simultaneous sequence non-specific end-labeling
and fragmentation of double-stranded and single-stranded nucleic
acids is presented herein. The method uses redox-reactive
coordination complexes to fragment and label RNA and DNA. These
labeled nucleic acids were found highly effective for hybridization
with DNA microchips containing oligonucleotide probes. The invented
protocols which have been developed for the fragmentation and
labeling of RNA and DNA may also be used for the fragmentation and
labeling of DNA prior to microchip hybridization. The invented
protocols utilize radical generating moieties to provide two
reactions simultaneously, labeling and fragmentation, for both RNA
and DNA.
[0027] In the labeling reactions, fluorescent dye is incorporated
mostly to 3' and 5' ends of nucleic acid fragments. This end
labeling feature without excessive concomitant nucleobase
modifications renders fragments for use in recognizing and
differentiating short sequences containing a few mismatches.
Generally, the protocols are run at temperatures ranging from 30 C.
to 95 C. Operating the protocols at temperatures approaching the
boiling point of water, or in the presence of a denaturing agent
(such as urea formamide, or guanidine chloride), confers unfolding
of the nucleic acids. This unfolding facilitates the production of
high yields of labeling and fragmentation, while eliminating the
influence of two-dimensional (2-D) structure on the protocol. This
protocol is particularly advantageous when working with RNA
inasmuch as RNA often has complicated 2-D structure.
[0028] Specifically, the inventors have utilized oxidants, which
have free radical characteristics, to facilitate the labeling of
nucleic acids. The advantages of the radical-mediated labeling
methods are simplicity and high speed. In addition, the reactions
are run at any temperature selected below the boiling point of
water, and preferably from between 30.degree. C. and 95.degree.
C.
[0029] The chemical radical-producing agents serve as chemical
nucleases in the invented method to produce single-stranded breaks
in nucleic acid probes. A myriad of coordination complexes are
utilized in the invented method, including, but not limited to,
1,10-phenanthroline-Cu(II) (hereinafter referred to as OP-Cu),
bleomycin-Fe(III) (hereinafter referred to as BLM-Fe), EDTA-Fe,
ascorbic acid-Cu, methylene-blue-Cu, metallogporphyrins, and other
chemical nucleases.
[0030] These radical producing complexes generate
amine-hydrazide-nucleic acid crosslinking under anaerobic
conditions. For example, in the presence of hydrogen peroxide under
anaerobic conditions, the BLM-Fe complex catalyzes the formation of
free nucleic acid bases and the aldehyde form of deoxyribose at the
abasic site of the DNA backbone. The backbone typically undergoes
scission in the presence of alkali or amines.
[0031] Generally, the invention embodies a two step method for
labeling DNA and RNA molecules with compounds containing primary
amines. First, DNA or RNA is modified under anaerobic conditions
with hydrogen peroxide, a coordination complex, and chemical
nucleases. Under anaerobic conditions, hydrogen peroxide and the
nucleases produce free radicals which attack the nucleic acids,
resulting in the formation of free nucleic acid bases and the
aldehyde forms of ribose or deoxyribose (See Equation 1). NA + Rad
Na i ' .times. --CH = O .times. .fwdarw. NH 2 .times. -- .times. R
NaCNBH 3 .times. .times. NA i ' .times. --C--NH--R i + j '' .times.
NA j '' .times. NH .times. --R + e .times. NA + Na j ' Equation
.times. .times. 1 ##EQU1## wherein NA designates nucleic acid and
Rad.is the product of a chemical radical production, of the type
discussed supra. NH.sub.2--R represents a compound having at least
one primary amino group and a second group which facilitates the
attachment of a label (e.g. ethyl diamine) in an indirect labeling
protocol. Alternatively NH.sub.2--R represents a compound
comprising a fluorescent dye conjugated with primer containing a
primary amine in a direct labeling protocol. As such, exemplary
NH.sub.2--R candidates include, but are not limited to an
amino-derivative of fluorophores, or any label containing a primary
amino group. NaCNBH.sub.3 is a reducing agent, .SIGMA.Na.sub.i,
--CH.dbd.O depicts an intermediate nucleic-acid form containing the
aldehyde or ketone moiety, typically on the 5' carbon or on the
sugar (ribose) itself. .SIGMA.Na.sub.j, represents all other
nucleic acids not containing the aldehyde or ketone moiety.
.SIGMA..sub.j''NA.sub.j''NH--R represents moieties resulting from
the attachment of a primary amine compound to nucleic acid moieties
not containing aldehyde groups, and .SIGMA..sub.eNA represents
other modifications of the starter nucleic acid pool, but which are
not involved in the cross linking reaction, The reactive aldehyde-
or ketone-group on the DNA and RNA thus serves in the second step
of the method as the substrate for subsequent labeling
reactions.
[0032] In the second step of the method, a primary amine is
combined with the aldehyde- or ketone-group in a condensation
reaction to produce a Schiff base or amides. The Schiff base is
reduced and the product of this reduction step is labeled with a
desirable tag. Alternatively, and as depicted above in Equation 1,
the reduction and labeling step can be combined. The reduction
and/or labeling processes can be done in aerobic or anaerobic
conditions.
[0033] The invented method produces high yields of crosslinked
complexes. The method is effective independent of the nucleic acid
sequence or the two-dimensional structure of nucleic acids. The
same invented protocol can be utilized to label both DNA and RNA.
The resulting labeled products are effective probes in
hybridization experiments.
Reaction Chemistry Detail
[0034] FIGS. 1 and 2 depict the mechanisms for dye cross linking to
modified DNA and RNA, respectively. Hemiacetal, lactone, and
5'aldehyde are the common intermediates in the oxidative strand
scission of nucleic acids by radical-generating agents. These
intermediates appear after base elimination has occurred and they
may serve as cross-linking sites for primary amines in the invented
radical-mediated nucleic acid labeling procedure.
[0035] The H-5'abstraction pathway under both aerobic and anaerobic
conditions often results in the production of an oligonucleotide
5'-aldehyde (FIG. 1C). The aldehyde interacts with amines through
the formation of a Schiff base in the same manner as described for
the H-4' anaerobic pathway. (FIG. 1A). For this labeling reaction,
the presence of sodium cyanoborohydride in the reaction buffer or
immediate sodium cyanoborohydride treatment following Fe-EDTA
treatment is desirable for fast Schiff base reduction and
production of a stable covalent complex, such as Molecule number 8
of FIG. 1.
[0036] Radicals generated with such redox-active coordination
complexes as OP-Cu and Fe-EDTA effectively attack both DNA and RNA.
The treatment of identical RNA and DNA sequences with OP-Cu
complexes linked to carrier oligonucleotides shows that both the
cutting sites and the kinetics of fragmentation are similar for RNA
and DNA. In addition, OP-Cu effectively cross-links histones both
to ribooligonucleotides and to deoxyribooligonucleotides as well as
to DNA in bulk chromatin in vitro and in vivo. OP intercalates into
the minor grove of B-form DNA and as such OP-Cu cleaves dsDNA more
readily then ssDNA. Generally, for RNA, OP-Cu degrades loop regions
more quickly than duplex regions.
[0037] Another possible difference in the reaction of OP-Cu with
DNA and RNA is that the punitive intermediate as depicted in FIG.
2, (Molecule No. 9) suggested for the RNA H-1' abstraction pathway
is a candidate for crosslinking with primary amines, resulting in
the formation of stable products.
[0038] The inventors have found that radical mediated labeling
seems to be an effective method for placing the majority of the dye
on the ends of the nucleic acid fragments. Radical mediated
labeling results in the crosslinking of the fluorescent dye to the
5'- or 3'-end of the nucleic acid strand. In addition, the
inventors found that in OP-Cu-mediated protein-DNA crosslinking,
the crosslinking occurs at the 5'-end or the 3'-end of the DNA
molecule in approximately 90% of the cross-linked complexes, and
crosslinking occurs randomly along the DNA fragment in
approximately 10% of the complexes.
[0039] In this detailed description, the radical-producing
complexes OP-Cu and Fe-EDTA are featured for illustrative purposes
only. As such, other radical producing complexes compatible with
preselected labels and target nucleic acids also are suitable.
[0040] The inventors have determined that redox-active coordination
complexes such as OP-Cu and Fe-EDTA can be effectively used for
sequence-dependent nucleic acid fragmentation and labeling with
fluorescent dyes as part of a DNA microchip protocol. Radicals
generated with OP-Cu and Fe-EDTA effectively attack both DNA and
RNA. RNA treated with the OP-Cu and the Fe-EDTA protocols was
highly suitable for hybridization with DNA microchips containing
oligonucleotide probes specific for the Bacillus group of
microorganisms. The inventors also demonstrated that both the OP-Cu
and the Fe-EDTA protocols were effective for the fragmentation and
labeling of DNA. Generally, OP-Cu and FeEDTA serve as
radical-generating chemical complexes.
[0041] As depicted in FIG. 1, A, five carbon atoms of the DNA sugar
residue have a total of seven hydrogen atoms available for
abstraction by an oxidizing agent. The main pathway of DNA cleavage
by OP-Cu is H-1 abstraction. OP-Cu also cleaves DNA with H-4
abstraction. OP-Cu degradation is associated with some slight
sequence specificity.
[0042] The Fe-EDTA complex is negatively charged and so does not
interact directly with the DNA molecule. Instead, the Fe-EDTA
complex, in the presence of hydrogen peroxide, produces hydroxyl
radicals (OH.) which have no charge and are therefore able to
diffuse into the molecule. Abstraction of the H-4 and H-5 are the
predominant pathways. Preference for individual hydrogen atoms was
H-5>H-4>H-2=H-3>H-1.
[0043] H-4 abstraction under anaerobic conditions results in
nucleobase release with the production of a hemiacetal intermediate
(FIGS. 1A, 1) that is in equilibrium with the aldehyde form of
deoxyribose (FIG. 1A, 2). Anaerobic conditions were utilized to
optimize amine cross-linking. Generally, oxygen was reduced in
reactants and reactant solutions by bubbling with argon. The
inventors found that, at least for Op-Cu oxidation protocols, a 15
percent increase in hybridization signal was realized when
anaerobic conditions were utilized.
[0044] The aldehyde group generated by the initial oxidation step
is attacked by a nucleophilic moiety (such as a primary amine or a
hydrazide), creating a reversible covalent bond (Schiff base). The
resultant imine undergoes spontaneous conversion with the
3'phosphodiester bond cleaved by the mechanism of
.beta.-elimination. This facilitates the simultaneous cross-linking
of amine or hydrazine derivatives of the fluorescent dyes to the
modified DNA at the same time as fragmentation occurs.
[0045] After fragmentation and cross-linking, reduction of the
Schiff base with sodium cyanoborohydride is desirable for
production of the final labeled product, (FIGS. 1A, 3). This
prevents removal of the cross-linked dye by
.delta.-elimination.
[0046] Another DNA intermediate used for labeling with
amino-derivatives of fluorescent dyes is meta-stable lactone in
FIG. 1B. Reaction of this lactone with a primary amine leads to two
labeled products, (FIGS. 1B, 5, 6).
[0047] The H-5' abstraction pathway under both aerobic and
anaerobic conditions results in the production of an
oligonucleotide 5'-aldehyde, as depicted in FIG. 1, C. In one
scenario, the aldehyde reacts with amines through the formation of
a Schiff base in the same manner as described for the anaerobic
pathway depicted in FIG. 1A. In this labeling reaction, the
presence of sodium cyanoboro-hydride in the reaction buffer or
immediate sodium cyanoborohydride treatment following Fe-EDTA
treatment is desirable for fast Schiff base reduction and
subsequent production of a stable covalent complex 8.
[0048] FIG. 2 depicts differences in labeling protocol between DNA
and RNA. Specifically, the presence of the hydroxyl group in the
2'-position of ribose results in the production of a putative
intermediate, (FIGS. 2A, 9) instead of lactone 4 (of FIGS. 1B, 4)
produced in the DNA manipulation. This lactone is able to react
with primary amines to form an amide (FIGS. 2A, 10), or a Schiff
base with an aldehyde group. The Schiff base then can be reduced to
produce a stable complex (FIGS. 2A, 11). The putative intermediate
(FIGS. 2B, 9) serves as a substrate for cross-linking with primary
amines to form stable labeled products (FIGS. 2A, 12).
[0049] The inventors have found that linking the dye to the end of
the nucleic acid fragment is more useful than having the dye
randomly localized along the fragment. Having the dye at the end of
the fragment causes minimal steric interference during subsequent
hybridization. The invented method of using radical mediated
labeling is an effective method for placing the majority of the dye
on the ends of the nucleic acid fragments.
[0050] In summary, radical mediated labeling results in the
cross-linking of the flourescent dye to the 5'- or 3'-end of the
nucleic acid strand, as depicted in FIGS. 1 and 2.
[0051] Generally, two protocols, direct and indirect, have been
developed to facilitate the fragmenting and labeling of nucleic
acids. Both protocols can be utilized with a broad spectrum of
derivatives of fluorescent dyes and at a wide range of
temperatures. The high temperature of reaction, or alternatively
the possibility of using a high concentration of a denaturant such
as urea, make the labeling-fragmentation reaction non-dependent
from the two-dimensional structures of the subject nucleic acid,
while also producing a high yield of reaction.
[0052] A schematic representation of the direct protocol is as
follows: NA--C.dbd.O+Label+Radical wherein NA--C.dbd.O represents a
nucleic acid with an aldehyde moiety attached thereto.
[0053] A schematic representation of the indirect protocol is as
follows: [0054] a) NA--C.dbd.O+Amine-containing
Intermediate+Radical; [0055] b) NA--C-Amine-containing
moiety+Label.
[0056] For the direct labeling protocol, the active aldehyde,
lactonic, or oxicarbomide groups produced within the sugar moiety
may be directly cross-linked with amine or hydrazine conjugates of
fluorescent dyes. The fluorescent dye Lissamine rhodamine B
ethylenediamine (LissRhod) was used for direct labeling of both RNA
and DNA. The resultant Schiff base was subsequently reduced with
sodium cyanoborohydride or sodium borohydride.
[0057] In the first stage of the indirect labeling protocol, a
compound containing a primary amine and another reactive moiety to
accommodate labels (e.g. (EDA) is cross-linked to the nucleic acids
instead of the fluorescent dye, forming a Schiff base. In the next
stage, the amino-modified nucleic acid may be cross-linked to
fluorophores containing amino-reactive groups, such as sulfonyl
chlorides, isothiocyanates, succinimidyl conjugates, fluorescamine,
aromatic dialdehydes (such as OPA, NDA, or ADA) or ATTO-TAG
reagents. For the indirect labeling protocol, Texas Red sulfonyl
chloride (TexRed) was used for labeling both RNA and DNA. The
indirect labeling protocol is especially useful for dyes that are
unstable in the presence of radicals, since the labeling step
occurs after the radical fragmentation reaction has been
completed.
Aerobic and Anaerobic Environment Detail
[0058] The proposed mechanisms of DNA degradation via hydrogen atom
abstraction can be influenced by the presence of oxygen. Most of
these reactions, with the exception of H-4' abstraction under
aerobic conditions, result in nucleobase release with the formation
of intermediates which may react with primary amines and thus may
be used for DNA crosslinking with aminoconjugates of fluorescent
dyes (Scheme 1). Alternatively, these intermediates may also be
cross linked with EDA and subsequently labeled with amino reactive
fluorophores.
[0059] H-4'abstraction under anaerobic conditions results in
nucleobase release with the production of a hemiacetal intermediate
that is in equilibrium with the aldehyde form of deoxyribose
(Molecule 2, FIG. 1A). The aldehyde group may be attacked by a
nucleophilic moiety (such as a primary amine or a hydrazide),
creating a reversible covalent bond (Schiff base), and the
resultant imine undergoes spontaneous conversion in which the
3'-phosphodiester bond is cleaved by the mechanism of
b-elimination. In this way, the crosslinking of amine or hydrazine
derivatives of the fluorescent dyes to the modified DNA can occur
at the same time as the fragmentation. After the fragmentation and
crosslinking, reduction of the Schiff base with sodium
cyanoborohybride is desirable for production of a stable covalent
bond, thus preventing removal of the cross-linked dye by
.beta.-elimination.
[0060] In contrast to the H-4'anaerobic pathway, H-4' abstraction
under aerobic conditions leads to the complete splitting of
deoxyribose, and the intermediates of this pathway may not be used
for labeling with aminoconjugates. Because the proposed
H-4'abstraction pathways indicate that amine crosslinking might be
less effective under aerobic conditions, anaerobic conditions were
used in this study.
[0061] To achieve anaerobic conditions, oxygen levels in all
reactants and reaction solutions can be reduced by bubbling with
neutral fluid (e.g. a noble gas). The OP-Cu and Fe-EDTA direct
protocols were run both with and without argon bubbling using
Bacillus cereus bulk RNA. The results indicated that argon bubbling
had little effect in the OP-Cu fragmentation process and had no
impact on fragmentation for the Fe-EDTA protocols (FIG. 6B). Also,
for the OP-Cu protocol, argon bubbling resulted in a 15% increase
in hybridization signal. Therefore, using argon bubbling with the
OP-Cu procedure will give optimal results. However, depending on
the application, a 15% loss in signal may be acceptable to reduce
the complexity and time of the procedure. If a small loss in signal
is acceptable, then removal of the argon bubbling is an option for
the OP-Cu procedures. For the Fe-EDTA protocol, the results were
reversed. For Fe-EDTA, removal of argon bubbling resulted in a 14%
increase in hybridization signal.
[0062] Both the OP-Cu and the Fe-EDTA protocols were ran using 16S
rDNA, which was produced by PCR amplification of bulk DNA from
Bacillus cereus 3329. The OP-Cu protocol was run in the manner
described supra for both the direct and indirect protocols for RNA.
Direct labeling-fragmentation reaction was performed with 15 mM OP,
1.5 mM Cu,100 mM H.sub.2O.sub.2, 1 mM LissRhod at 45.degree. C. for
30 min under argon bubbling and followed by reduction by 20 mM
NaCNBH.sub.3. Indirect reaction was carried out with 1.5 mM OP,
0.15 mM Cu, 10 mM H.sub.2O.sub.2, 50 mM EDA at 45.degree. C. for 30
min under argon bubbling, followed by reduction by 20 mM
NaCNBH.sub.3, and labeled with 12.5 mM TexRed.
RNA/DNA-labeling with OP-Cu
[0063] OP-Cu binds to double stranded DNA in the minor groove, and
in the presence of hydrogen peroxide, promotes DNA cleavage by the
abstraction of a hydrogen atom. Five carbon atoms of the DNA sugar
residue have a total of seven hydrogen atoms which are available
for abstraction by an oxidizing agent. The main pathway of DNA
cleavage by OP-Cu is H-1' abstraction, but OP-Cu can also cleave
DNA by a minor pathway that begins with abstraction of H-4'. The
degradation of DNA by OP-Cu has some slight sequence
specificity.
[0064] Treatment of identical RNA and DNA sequences with OP-Cu
complexes linked to carrier oligonucleotides has demonstrated that
both the cutting sites and the kinetics of fragmentation are
similar for RNA and DNA.
[0065] A myriad of different concentrations, temperatures and
reaction times are suitable to run the protocol. Suitable
concentrations of all reagents range from 0.01 mM to 1000 mM at
temperatures ranging from 10.degree. C. to 100.degree. C. Exemplary
reactant concentrations, temperatures and times are illustrated in
Table 1, infra. Generally however, OP concentrations of between 1.5
and 15 mM and copper concentrations of from 0.15 and 1.5 were
suitable. H.sub.2O.sub.2 concentrations of between 10 and 100 mM
also provided good results. Temperatures of between 30.degree. C.
and 45.degree. C. produced good results.
[0066] The inventors found that the Op-Cu protocol was highly
effective for RNA fragmentation and labeling when run at
temperatures below the boiling point of water (i.e. 100.degree.
C.), preferably between 45.degree. C. and 100.degree. C. and most
preferably at approximately 95.degree. C. Reaction times will vary
from a few seconds to several hours depending on temperature. For
example, with a reaction temperature of 95.degree. C., a reaction
time of approximately 1-2 minutes is all that is required. With
reaction temperatures of approximately 45.degree. C., a 30 minute
reaction time may be required. A reaction temperature of 0.degree.
C. will require a reaction time of approximately 2 hours. In light
of the foregoing, for some applications, such as field applications
where energy input is a consideration, a low reaction temperature
can be an advantage.
Indirect OP-Cu Protocol
[0067] A first stage in the indirect labeling protocol is a cross
linking of ethylenediamine (EDA) to the nucleic acids, instead of
to the fluorescent dye. This produces the condensation product,
i.e., the Schiff base. Next, the amino-modified sugar is
fluorescently labeled with sulfonyl chlorides, isothiocyanates,
succinimidyl conjugates, fluorescamine, aromatic dialdehydes such
as OPA, NDA, ADA or ATTO-TAG reagents.
[0068] Texas Red sulfonyl chloride (TexRed) was used for indirect
labeling of both RNA and DNA.
[0069] The indirect labeling protocol is particularly useful for
dyes that are unstable in the presence of radicals, since the
labeling step occurs after the radical fragmentation reaction has
been completed.
[0070] Electrophoresis of RNA indirectly labeled under denaturing
conditions revealed that increasing the OP-Cu and hydrogen peroxide
concentrations resulted in a decrease in RNA length. A suitable
condition for indirect labeling was treatment with 1.5 mM OP/0.15
mM Cu/10 mM H2O2 and heating at 45.degree. C. for 30 minutes. RNA
fragments between 50 b and 100 b are produced with this protocol.
Clearly defined hybridization signals also result.
[0071] FIG. 3 shows hybridization of a microchip with bulk B.
thuringiensis RNA fragmented and labeled via the OP-Cu indirect
labeling protocol. The label used was TexRed. The numbers in the
figure indicate the probes listed in Table 1.
[0072] Screening of several different reactant concentrations
demonstrated that a preferred protocol for indirect labeling with
OP-Cu includes concentrations of 1.5 mM OP/0.15 mM Cu/10 mM
H.sub.2O.sub.2, at 45.degree. C. for 30 minutes. These
concentrations resulted in the strongest hybridization signal
(Table 3, treatment 3 and FIG. 3, c) and RNA fragments between
approximately 50 and 100b in length (FIG. 4A, c). Increasing the
hydrogen peroxide concentration 10 times to 100 mM resulted in a
decrease in the hybridization signal (Table 3, treatment 2 and FIG.
3b) and a decrease in the RNA length (FIG. 4A, b). Increasing the
concentrations of all reagents 10 times to 15 mM OP/1.5 mMCu/100 mM
H.sub.2O.sub.2 resulted in almost complete degradation of the RNA
(FIG. 4A, a) and a further decrease in the hybridization signal
(FIG. 3a and Table 3, treatment 1).
[0073] There are some unexpected differences in the reactions of
OP-Cu with DNA and RNA. For example, because OP intercalates into
the minor groove of B-form DNA, OP-Cu cleaves dsDNA more
efficiently then ssDNA. However, for RNA, OP-Cu degrades loop
regions more quickly than duplex regions. This difference in the
reactions for DNA and RNA may be due to steric effects. The
protocol utilized for OP-Cu treatment is based on that disclosed
for protein-nucleic acid cross-linking, in S. G. Bavykin et al.
Anal. Biochem., (1998) 263, 26-30, and incorporated herein by
reference.
[0074] As noted supra, OP-Cu binds to double stranded DNA in the
minor groove. In the presence of hydrogen peroxide, OP-Cu promotes
cleavage by the abstraction of a hydrogen atom. The main pathway of
DNA cleavage by OP-Cu is H-1 abstraction. OP-Cu also can cleave DNA
via H-4 abstraction (See FIGS. 1A, 1).
Direct OP-Cu Protocol
[0075] In the direct labeling protocol, nucleic acid treatment of
OP-Cu results in the production of active aldehyde, lactonic, or
oxicarbomide groups within the sugar moiety. These groups are then
directly cross-linked with amine or hydrazine conjugates of
fluorescent dyes in a condensation step. The resultant Schiff base
is subsequently reduced with a suitable reducing agent. Exemplary
reducing agents include, but are not limited to, sodium
cyanoborohydride and sodium borohydride.
[0076] In order to optimize the direct OP-Cu protocol, experiments
with different concentrations of reactants were conducted with
variations in temperature and time of reaction. Reaction times of
between 10 and 30 minutes are appropriate. Exemplary reaction
concentrations, temperatures and times are illustrated in Table 1.
Results of the invented labeling method were compared with the
results obtained using the Magnesium-Sodium Periodate method
discussed supra. Strongest hybridization signals were obtained with
1.5 mM OP, 0.15 mM Cu, and 10 mM H.sub.2O.sub.2 at 95.degree. C.
for 30 minutes, and also when concentrations were 15 mM OP, 1.5 mM
Cu, and 10 mM H.sub.2O.sub.2 at 95.degree. C. for 10 minutes.
[0077] For the direct OP-Cu labeling procedure, screening
experiments (Table 1, FIG. 5) demonstrated that the optimal
reactant concentrations were 15 mM OP/1.5 mM Cu/100 mM
H.sub.2O.sub.2. These concentrations produced a strong
hybridization signal. FIG. 5 shows the effect of urea on direct
OP-Cu and FeEDTA labeling-fragmentation of RNA.
[0078] To determine if the OP-Cu reaction could be run more
quickly, experiments were conducted with variations in temperature
and time of reaction (Table 5). The strongest hybridization signals
were obtained with 1.5 mM OP/0.15 mM Cu/10 mM H.sub.2O.sub.2at
95.degree. C. for 30 min. and with 15 mM OP/1.5 mM Cu/10 mM
H.sub.2O.sub.2 at 95.degree. C. for 10 min. (Table 5, treatment 4
and 7). Thus, the OP-Cu reaction can be shortened to 10 minutes if
the temperature is raised to 95.degree. C.
[0079] A variation of the OP-Cu protocol was run in which the
reductant NaCNBH.sub.3 was included in the reaction step, as
depicted in FIG. 1A. This contrasts to the standard protocol
wherein the reduction step is carried out after the scission
reaction has been completed. The inclusion of the reductant in the
reactant step produced an equivalent level of both fragmentation
and hybridization signal to the invented protocol. However, this
combining of scission and reduction procedures reduced the time for
fragmentation and labeling by 30 minutes.
[0080] An exemplary procedure to effect a direct OP-Cu protocol is
as follows: To maintain anaerobic conditions, all reagents were
bubbled with argon for 15 seconds before use, and the reaction
solutions were bubbled with argon for 15 seconds between each step.
Total reaction volume was 100 .mu.l. RNA (10 or 20 .mu.g), 20 .mu.l
of 100 mM sodium phosphate (pH=7), 7M urea, and DEPC treated
H.sub.2O were combined and bubbled with argon. After addition of
o-phenanthroline hydrochloride monohydrate (OP) (Fluka, Ronkonkoma,
N.Y.), CuSO.sub.4.times.5 H.sub.2O (Cu), and 1 .mu.l of 100 mM
LissRhod, solution was bubbled with argon and preheated for 3
minutes. The solution was again bubbled with argon and
H.sub.2O.sub.2was added. The reaction solution was then bubbled
with argon and heated for 10 to 30 min. Reaction was stopped by
addition of 2 .mu.l 0.5M EDTA and incubation in a room temperature
water bath for 1 min. Reduction was carried out by addition of
sodium cyanoborohydride to 20 mM and incubation at room temperature
in the dark for 30 min. RNA was precipitated in 96% ethanol/0.4M
sodium acetate at -80.degree. C. for 20 min. After centrifugation
at 14,000 rpm for 5 minutes, RNA pellets were washed twice with
ethanol. Excess LissRhod was removed from RNA by butanol treatment
as described above and RNA pellets were suspended in 10 to 20 .mu.l
DEPC H.sub.2O. TABLE-US-00001 TABLE 1 Comparison of Hybridization
Signals of Direct and Indirect RNA labeling via Mg2+, OP-Cu, and
Fe-EDTA Methods Reaction Conditions Hybridization Temp Time
Reactant Signal Method (.degree. C.) (min) Concentrations
(u/.mu.g/sec) Mg.sup.2+ Direct 95 40 60 mM Mg.sup.2+ 844 Mg.sup.2+
Indirect 95 40 60 mM Mg.sup.2+ 1142 Fe-EDTA Direct 95 10 1.5 mM 870
Fe-EDTA 10 M H.sub.2O.sub.2 Fe-EDTA Indirect 95 10 1.5 mM 1094
Fe-EDTA 1 mM H.sub.2O.sub.2 1 mM H.sub.2O.sub.2 OP-Cu Direct 45 30
15 mM OP 949 1.5 mM Cu 100 mM H.sub.2O.sub.2 OP-Cu Indirect 45 30
1.5 mM OP 1309 0.15 mM Cu 10 mM H.sub.2O.sub.2
[0081] The fluorescent dye Lissamine rhodamine B ethylenediamine
(LissRhod) was utilized in the direct labeling of both RNA and
DNA.
[0082] For direct labeling of RNA, 10 times higher OP-Cu
concentrations than that used in the indirect protocol produced the
same average RNA lengths as produced by the indirect method. This
difference was probably due to the lesser amounts of amine utilized
in the direct protocol versus the indirect labeling protocol.
[0083] The optimal direct and indirect OP-Cu protocols resulted in
identical hybridization patterns (FIG. 5) and both produced strong
hybridization signals. Optimal reactant concentrations for the
direct labeling protocol are 10 times higher than the optimal
concentrations for the indirect labeling protocol.
[0084] The quantum yield of TexRed conjugates has been found to be
higher than the quantum yield of LissRhod conjugates. The indirect
protocol includes a 50 fold higher concentration of the amine
group, and it allows the fluorophore a much longer time to
crosslink (overnight as compared to 30 minutes).
[0085] The inventors found a resistance of double stranded regions
to fragmentation, thereby making it difficult to obtain strong
hybridization signals for probes complementary to certain hairpin
regions of RNA and DNA. In these instances, urea was utilized to
eliminate the influence of certain three-dimensional structures
(such as hairpins) on the labeling-fragmentation procedure and to
improve the signal for probes complementary to hairpin regions. The
inventors found that the addition of urea to the invented protocol
increases the sensitivity of the double stranded regions within the
RNA molecule to fragmentation.
[0086] Surprisingly and unexpectedly, the inventors found that when
other reagents are held constant, the addition of urea dramatically
increased the sensitivity of RNA to hydrolysis with OP-Cu. For
example, when 3.5 M-7.0 M urea was included in the reaction and the
hydrogen concentration was lowered 10-fold to 10 mM, the same
degree of RNA fragmentation and a higher hybridization signal was
obtained, compared to when higher levels of hydrogen peroxide and
no urea is utilized.
RNA/DNA Labeling with Fe-EDTA Detail
[0087] An Fe-EDTA radical generating system was employed for a
sequence-nonspecific labeling method. The Fe-EDTA complex is
negatively charged and thus does not interact directly with the DNA
molecule. Instead, the Fe-EDTA complex, in the presence of hydrogen
peroxide, produces hydroxyl radicals which have no charge and which
therefore are able to diffuse into the DNA molecule. Hydroxyl
radicals are able to abstract any of the hydrogen atoms from the
carbon atoms within the deoxyribose residues of B-form DNA, but
abstraction from the 4' and 5'-positions are the predominant
pathways. Preference for individual hydrogen atoms was found to be
H-5'>H-4'>H-2'=H-3'>H-1', which correlates with the
accessibility of the individual hydrogen atoms to a solvent.
[0088] As with the OP-Cu method, a direct and an indirect labeling
protocol were used to label DNA and RNA. Generally, exemplary
Fe-EDTA protocols are found in M. A. Price et al. Methods Enzymol,
202, 194-219, Marshall, et al., Biochemistry 20, 244-250 and
Tullius et al., Methods Enzymol. 155, 537-559, all incorporated
herein by reference. Suitable results were obtained at 95.degree.
C. for 10 minutes.
[0089] The indirect Fe-EDTA protocol that gave the strongest
hybridization signal was 1.5 mM Fe/10 mM H.sub.2O.sub.2/1 mM NaAsc
(Table 4, treatment 6). The optimal indirect Fe-EDTA protocol
required a 10 fold lower concentration of H.sub.2O.sub.2 than the
direct protocol. PAGE gel electrophoresis data indicated that the
optimal indirect Fe-EDTA protocol resulted in less fragmentation
(FIG. 5A g) than the optimal direct Fe-EDTA protocol (FIG. 5A c).
This result is consistent with the results for the Op-Cu system, in
which the indirect protocol also required lower reactant
concentrations and less fragmentation than the direct protocol. As
was discussed above, this result may be due to differences in the
fluorophores or differences in the direct and indirect
protocols.
Fe-EDTA Direct Protocol
[0090] To maintain anaerobic conditions, all reagents are bubbled
with a neutral fluid (such as argon, helium, or other relatively
unreactive gases) before use, and the reaction solutions were
bubbled with the fluid between each step. The Fe-EDTA complex
consists of 0.5M EDTA and 0.25M ammonium iron (II) sulfate. The
following concentrations are for illustrative purposes only
inasmuch as commercial scales are considerably larger. Also,
reaction times and temperatures may vary to take into consideration
batch processing effects and the like.
[0091] In the laboratory scaled protocol, total reaction volume was
100 .mu.l. RNA (10 or 20 .mu.g), 20 .mu.l 100 mM sodium phosphate
(pH=7), DEPC treated H.sub.2O, 7M urea, and the Fe-EDTA complex
were combined and bubbled with argon. After bubbling, 1 .mu.l of
100 mM LissRhod was added and solution was bubbled with argon and
preheated for 3 minutes at 95.degree. C. H.sub.2O.sub.2 and sodium
ascorbate (NaAsc) were added. The reaction solution was again
bubbled with argon and then heated to 95.degree. C. for 10 to 30
min. Reaction was stopped by addition of 10 .mu.l 1M thiourea and
incubation in a room temperature water bath for 1 min. Reduction
was carried out by addition of 10 .mu.l of 200 mM sodium
cyanoborohydride and incubation at room temperature in the dark for
30 min. Labeled RNA was precipitated in 96% ethanol/0.4M sodium
acetate at -80.degree. C. for at least 20 min. After centrifugation
at 14,000 rpm for 5 minutes, RNA pellets were washed twice with
ethanol. Excess LissRhod was removed by butanol treatment as
described above. RNA pellets were suspended in 10 to 20 .mu.l DEPC
H.sub.2O.
Indirect Labeling Protocol
[0092] As in the direct protocol above, the following
concentrations are provide for illustrative purposes only.
Commercial scale operations obviously require larger volumes and
typical processing requirements.
[0093] In the laboratory protocol, for indirect labeling, LissRhod
is replaced in the fragmentation protocol by 10 .mu.l 0.5M
ethylenediamine (EDA). After precipitation, the RNA pellet was
dissolved in 60 .mu.l or 100 .mu.l 100 mM sodium carbonate (pH
9.0). The mixture was then transferred to an ampule containing
Texas Red sulfonyl chloride (TexRed) (Molecular probes, Eugene,
Oreg.), precooled with ice, and incubated on ice overnight. The
reaction was stopped by adding 25 .mu.l or 40 .mu.l 1M acetic acid
and the mixture was diluted with 200 .mu.l 100 mM sodium acetate
(pH 4). Excess TexRed was removed from RNA by butanol treatment as
described above. RNA pellets were suspended in 10 to 20 .mu.l DEPC
H.sub.2O.
[0094] Screening of several different reaction conditions
demonstrated that the direct Fe-EDTA protocol that gave the highest
hybridization signal was 1.5 mM Fe/10 mM H.sub.2O.sub.2/10 mM NaAsc
(Table 1). PAGE Gel Electrophoresis demonstrated that this reaction
condition produced RNA fragments between approximately 50 and 100b
in length (FIG. 5A. c). Increasing the concentration of Fe to 15 mM
resulted in an increase in the length of RNA (FIG. 5A, b) and a
decrease in the hybridization signal (Table 4, treatment 1). As
such, the inventors found that Fe inhibits fragmentation, which is
resulting in a decrease in the hybridization signal. Increasing the
level of H.sub.2O.sub.2 in addition to increasing the level of Fe
resulted in almost complete degradation of the RNA (FIG. 5A, d) and
a further decrease in the hybridization signal (Table 4, treatment
3).
Nucleic Acid Isolation and Preparation Detail
[0095] In one protocol, chip-attached probes specific for the
Bacillus group of microorganisms were effectively hybridized with
nucleic acids, which were labeled via the invented protocol.
[0096] RNA was isolated from frozen cell pellets of Bacillus cereus
9620, Bacillus cereus 3329, and Bacillus thuringiensis 4042B.
Bacillus thuringiensis 4042B was used as a mimic of Bacillus
antracis, as both have identical 16S rRNA sequences. Cells were
lysed via standard bead beating protocol such as that disclosed in
Sambrook et al., Molecular Cloning, A Laboratory Manual, 2.sup.nd
Ed. CSH (1989) and incorporated herein by reference. RNA was
isolated by phenol extraction and precipitated by addition ammonium
acetate and ethanol. Surprisingly and unexpectedly, the inventors
found that this precipitation protocol allowed for the RNA to be
stored at -80.degree. C. overnight, without sustaining damage.
[0097] After centrifugation at 14,000 rpm for 5 minutes, RNA
pellets were washed with ethanol, and suspended in water containing
an RNAse inhibitor. One such exemplary inhibitor is DEPC (diethyl
pyrocarbonate).
[0098] With regard to DNA preparation, 16S rDNA was synthesized by
PCR amplification of bulk B. cereus 9620 and B. anthracis AMES DNA
polymerase (available from Ambion, Austin, Tex.) using 11F and 1512
primers.
[0099] Two protocols, enumerated in the examples below have been
developed. Both were successfully used for B. medusa labeling and
fragmentation at the same time.
EXAMPLE 1
[0100] To assure anaerobic conditions, and just before the start of
the reactions, all solutions are bubbled with argon. Ethylene
diamine (EDA) is included in the initial reaction mixture in the
presence of hydrogen peroxide and OP-Cu or EDTA-Fe. This initial
step has a duration of approximately 30 minutes and occurs at
approximately 45.degree. C. The EDA reacts with the aldehyde groups
of the DNA or RNA to form a Schiff base.
[0101] The labeling process is continued in the same reaction
vessel by reducing the double bond in the base with a reducing
agent such as sodium cyanoboro-hydride. This step has a duration of
approximately 30 minutes and takes place at room temperature. The
Schiff base is then reprecipitated with acetone. The product of
this reduction is then labeled by adding Texas Red sulfonyl
chloride (TexRed-SuCl) and subsequently hybridized with a
microchip.
EXAMPLE 2
[0102] Anaerobic conditions are established as in Example 1, above.
In this example, nucleic acids were modified with OP-Cu or EDTA-Fe
in the presence of hydrogen peroxide and amino-dye Lissamine
rhodamine B ethylenediamine (LissRH-EDA) over a period of
approximately 30 minutes at 45.degree. C.
[0103] The resulting LissRH-EDA-nucleic acid Schiff base was
reduced with sodium cyanoborohydride in the same reaction vessel
over a period of 30 minutes at room temperature. Alternatively,
crosslinking of the dye and Schiff base reduction were integrated
in one step.
[0104] The DNA microchips utilized in this study consisted of an
array of polyacrylamide gel pads affixed to a glass slide. These
gel pads served as three-dimensional supports for the
immobilization of oligonucleotide probes. This DNA microchip
technology requires random fragmentation and fluorescent labeling
of target nucleic acids prior to hybridization. Nucleic acid
fragmentation is necessary to reduce the size of the nucleic acid
molecules so that they can easily enter the pore spaces of the
polyacrylamide gel pads and access the oligonucleotide probes.
Nucleic acid fragmentation also allows different regions of the
target nucleic acid molecule to hybridize independently to each of
the immobilized oligonucleotides.
[0105] Fluorescent labeling of target nucleic acids is required for
hybridization detection. Although fluorescent labeling is less
sensitive than radioactive labeling, fluorescent labeling offers
several advantages. Fluorescent dyes do not pose radiation hazards,
and thus their use and disposal is less problematic. In addition,
fluorescent labels can be detected in real time with high
resolution. Treatment of DNA or RNA with an oxidizing agent or free
radical results in the production of a reactive abasic site on the
nucleic acid molecule. This sort of modification protocol obtained
after purine methylation is disclosed in Pruss, D. and Bavykin, S.
G. (1997) Methods, 12, 36-47 and Pruss, D., Gavin, I. M., Melnik,
S., and Bavkin, S. G. (1999) Methods Enzymol., 304, 516-533, and
incorporated herein by reference. Nucleic acid treatment with
radical-generating coordination complexes is also disclosed in
Gavin, I. M., Melnik, S. M., Yurina, N. P., Khabarova, M. I., and
Bavykin, S. G. (1998) Anal. Biochem., 263, 26-30 and incorporated
herein by reference, for crosslinking of proteins to nucleic acids
through the amino groups or through the imidazole rings of
histidines. This reactive abasic site is useful for the direct
crosslinking of nucleic acids to fluorophores containing amino
groups, or for the indirect crosslinking of nucleic acids to amino
reactive fluorophores through ethylenediamine (EDA). The inventors
in the present invention utilize the radical generating systems to
both fragment nucleic acids and in the same reaction produce
reactive sites that would allow the crosslinking of the nucleic
acid fragments to fluorophores.
RNA Isolation
[0106] Total RNA was isolated from frozen cell pellets of Bacillus
cereus (str. 9620), and Bacillus thuringiensis (str. 4042B). Cells
were lysed via standard bead beating protocol, as outlined supra.
RNA was isolated by phenol extraction and precipitated by addition
of 0.5 volumes of 7.5M ammonium acetate and 2.5 volumes of ethanol
and storage at -80.degree. C. overnight. After centrifugation at
14,000 rpm for 5 minutes, RNA pellets were washed with 80% ethanol,
and suspended in DEPC (diethyl pyrocarbonate) treated H.sub.2O.
DNA Preparation
[0107] 16S rDNA was synthesized by PCR amplification of bulk DNA
from Bacillus cereus 3329 with AmpliTaq DNA polymerase (Ambion,
Austin, Tex.) using 11F and 1512R primers.
Comparison of Methods
[0108] To determine the effectiveness of the OP-Cu and Fe-EDTA
systems, the hybridization signals obtained with these systems to
the hybridization signal were compared with signals obtained with a
Magnesium-Sodium Periodate labeling and fragmentation method
developed by the inventors. Direct labeling and indirect labeling
variations were ran using all three methods using Bacillus cereus
9620 bulk RNA. All three methods gave identical hybridization
patterns and the hybridization signals for all three methods were
approximately equivalent (FIG. 5 and Table 1).
[0109] In all three systems, indirect labeling produced 20 to 30%
higher hybridization signals than direct labeling. This result is
consistent with the results found for both the OP-Cu and Fe-EDTA
methods.
[0110] An exemplary protocol of the invented comparison method
follows herewith. It should be noted that the reaction volumes
utilized are relative to all reactants. For the sake of
illustration, specific reaction volumes are employed herein.
[0111] Given a total reaction volume of 100 .mu.l, RNA (20 mg) and
DEPC-treated H.sub.2O were combined and preheated at 95.degree. C.
for 5 minutes. MgCl.sub.2 was added to 60 mM and the reaction
solution was heated at 95.degree. C. for 40 minutes. Phosphatase
treatment was carried out by addition of 3 .mu.l 10.times. alkaline
phosphatase buffer (Promega, Madison, Wis.) and 0.2 .mu.l alkaline
phosphatase (1.mu./.mu.l) (Promega, Madison, Wis.) and heating at
37.degree. C. for 30 minutes. Oxidation was conducted by addition
of 6.5 .mu.l of 100 mM sodium periodate and incubation at room
temperature for 20 minutes. Labeling was carried out by addition of
3.5 .mu.l of 100 mM Lissamine rhodamine B ethylenediamine
(LissRhod) (Molecular Probes, Eugene, Oreg.) and 1.65 .mu.l of 1M
HEPES (pH 7.5) and heating at 37.degree. C. for 1 hour. Reduction
was conducted by addition of 6.7 .mu.l of 200 mM sodium
cyanoborohydride and incubation at room temperature for 30 minutes.
Labeled RNA was precipitated by addition of 15 volumes of 2%
lithium perchlorate in acetone and stored at -20.degree. C. for 20
min. After centrifugation at 14,000 rpm for 5 minutes, RNA pellets
were washed twice with acetone and dried at 55.degree. C. for 10
minutes.
Butanol Treatment
[0112] Excess LissRhod was removed from RNA by butanol treatment:
RNA pellets were suspended in 300 .mu.l DEPC treated H.sub.2O, and
samples were concentrated to 60 .mu.l by removal of water with
butanol. Treatment was repeated until butanol was free of color.
RNA was precipitated in 15 volumes of 2% LiClO.sub.4 in acetone at
-20.degree. C. for 20 min. After centrifugation at 14,000 rpm for 5
minutes, RNA pellets were washed twice with acetone, dried at
55.degree. C. for 10 minutes, and suspended in 10 to 20 .mu.l DEPC
treated H.sub.2O.
[0113] Fragmented and labeled RNA samples were analyzed by
polyacrylamide gel electrophoresis.
Genus- and Species-specific Oligonucleotide Probes.
[0114] For selection of genus-specific probes, the 16S rRNA
sequence from a specific microorganism belonging to the genus was
used to create a set of all possible 20b oligonucleotide probes
(the set consisted of L-19 oligonucleotides, where L denotes the
length of the entire 16S rRNA sequence). Each potential probe was
tested against all available 16S rRNA sequences (GenBank and RDP)
by a function that estimates the relative duplex stability
according to the number and position of mismatches. If the 16S rRNA
of any microorganism that did not belong to the genus of interest
formed stable duplexes with any oligonucleotide considered as a
probe for the microchip, this oligonucleotide was excluded from the
list of probes. A similar procedure was carried out for the
selection of species-specific probes. A final set of 15
oligonucleotide probes each approximately 20b in length (Table 2)
was selected for application to the DNA microchip.
[0115] The selected oligonucleotides (Table 2) were synthesized on
an automatic DNA/RNA synthesizer (Applied Biosystems 394) using
standard phosphoramide chemistry. A 5'-Amino-Modifier C.sub.6 (Glen
Research, Sterling, Va.) was linked to the 5'-end of the
oligonucleotides. TABLE-US-00002 TABLE 2 Oligonucleotide probes on
DNA microchip 16S rRNA 5'-end Probe Length Sequence location Target
1 17 ACG GGC GGT GTG TRC AA 1400 Universal 2 18 GWA TTA CCG CGG CKG
CTG 529 Universal 3 18 TGC CTC CCG TAG GAG TCT 345 Eubacteria 4 17
ACC GCT TGT GCG GGC CC 938 Eubacteria 5 20 CGA AGC CGC CTT TCA ATT
TC 203 B. cereus Group 6 20 CAA CTA GCA CTT GTT CTT CC 455 B.
cereus Group 7 20 TGT CAC TCT GCT CCC GAA GG 1038 B. cereus Group 8
20 CGG TCT TGC AGC TCT TTG TA 1257 B. cereus Group 9 23 ATG CGG TTC
AAA ATG TTA TCC GG 175 B. cereus strs. 9620 and B. thuringiensis
str. 4042B 10 20 TTC GAA CCA TGC GGT TCA AA 186 B. cereus strs.
9620 and B. thuringiensis str. 4042B 11 20 TTC GAA CTA TGC AGT TCA
AA 186 B. mycoides str. 6462m 12 23 CAA TTT CGA ACT ATG CAG TTC AA
187 B. mycoides str. 6462m
[0116] A microchip consisting of an array of 100.times.100.times.20
.mu.m polyacrylamide gel pads affixed to a glass slide and spaced
100 .mu.m from each other was manufactured via photopolymerization
as disclosed in Gushin et al. Anal Biochem 250, 203-211 (1997) and
incorporated herein by reference. The gel pads were activated as
described in Proudnikov et al. Nucl. Acids Res 24, 4535-4542,
incorporated herein by reference. This resulted in the production
of aldehyde groups within the gel pads. Each oligonucleotide was
applied to a unique gel pad within the array and each gel pad
received a preselected volume (for example 6 nL) of the
oligonucleotide solution. The hybridization solutions and protocol
are disclosed in the references cited above. Generally, though, the
hybridization solution consisted of DEPC treated H.sub.2O, 3M
GuSCN, 0.5M EDTA (pH 7.0), 1M HEPES (pH 7.5), and RNA solution. The
hybridization solution was filtered, and then heated at 95.degree.
C. for 3 min before placed on ice. The hybridization solution was
added to a hybridization chamber, and the hybridization chamber was
affixed to a microchip. The microchip was allowed to hybridize
overnight at room temperature in the dark. After hybridization, the
chamber and hybridization solution were removed from the microchip,
and the microchip was washed with NaCl, sodium phosphate at neutral
pH, EDTA, and Tween. After washing the microchip was imaged using a
fluorescence microscope, and CCD camera.
Analysis of Hybridization Data
[0117] The fluorescent intensity of each gel element was quantified
from the WinView image using LabView software. The score for each
gel element was calculated by subtracting the averaged fluorescent
intensity of the area immediately surrounding the gel element (i.e.
the background) from the averaged fluorescent intensity of the
entire area of the gel element. To compare experimental treatments,
the hybridization signal for each experimental treatment was
calculated by averaging the scores for the four oligonucleotide
probes targeting the anthracis group, probes 5 to 8 (Table 2). The
intensity of the hybridization signal was used to assess the
effectiveness of different reaction parameters for the
fragmentation and labeling procedures. Hybridization Tables 3-6
represent average signals calculated from data obtained in a single
experiment. Within each experiment, treatments were replicated from
2 to 4 times, and the variation in hybridization signals for each
treatment was less than 20%. TABLE-US-00003 TABLE 3 Indirect
fluorescent RNA labeling with OP-Cu* Concentrations (mM) Treat-
Nucleic Texas Hybridization*** ment Acid OP Cu H.sub.2O.sub.2 EDA
Red Signal (u/.mu.g/sec) 1 RNA** 15.0 1.50 100 50 20.0 50 2 RNA**
1.5 0.15 100 50 20.0 930 3 RNA** 1.5 0.15 10 50 20.0 1700 *All
fragmentation and labeling reactions were run for 30 min. at
45.degree. C. **B. thuringiensis 4042B bulk RNA ***Average of
hybridization signal from probes 5, 6, 7, 8.
[0118] TABLE-US-00004 TABLE 4 Direct method of fluorescent RNA
labeling with Fe-EDTA* Concentrations (mM) Treat- Nucleic Liss.
Hybridization*** ment Acid Fe H.sub.2O.sub.2 NaAsc rhod. Signal
(u/.mu.g/sec) 1 RNA** 15.0 10 1 1 55 2 RNA** 1.5 10 1 1 280 3 RNA**
150.0 100 1 1 14 4 RNA** 15.0 10 10 1 52 *All fragmentation and
labeling reactions were run for 10 min. at 95.degree. C. **B.
cereus 9620 bulk RNA ***Average of hybridization signal from probes
5, 6, 7, 8.
[0119] TABLE-US-00005 TABLE 5 Direct OP-Cu RNA* labeling: variation
in reaction parameters Treat- Temp Concentrations (mM)
Hybridization*** ment (.degree. C.) Time (min.) OP Cu
H.sub.2O.sub.2 Signal (u/.mu.g/sec) 1 45 30 15.0 1.50 100 650 2 70
30 15.0 1.50 50 200 3 70 30 1.50 0.15 10 475 4 95 30 1.50 0.15 10
1100 5 95 10 1.50 0.15 10 710 6 95 10 15.0 1.50 50 150 7 95 10 15.0
1.50 10 1150 8 95 10 1.5 0.15 50 500 9 95 10 1.5 0.15 10 760 *B.
cereus 9620 bulk RNA **Average of hybridization signal from probes
5, 6, 7, 8.
[0120] TABLE-US-00006 TABLE 6 Direct OP-Cu RNA* labeling** with
variations in urea concentration Concentrations (mM)
Hybridization*** Treatment Urea (M) OP Cu H.sub.2O.sub.2 Signal
(u/.mu.g/sec) 1 0.0 15.0 1.5 100 570 2 3.5 15.0 1.5 100 90 3 3.5
15.0 1.5 10 815 4 7.0 15.0 1.5 100 56 5 7.0 15.0 1.5 10 280 *B.
cereus 9620 bulk RNA **All fragmentation and labeling reactions
were run for 30 min. at 45.degree. C.
[0121] Another DNA intermediate that may be used for labeling with
aminoderivatives of fluorescent dyes is metastable lactone.
(Molecule number 4 in FIG. 1B) This lactone is an intermediate in
both aerobic and anaerobic H-1' abstraction pathways. Reaction of
this lactone with a primary amine leads to products depicted as
Molecules 5 and 6 in FIG. 1.
Denaturing Agent Detail
[0122] Optionally, denaturing agents are included in the reactions
to disrupt the secondary structure of the 16S rRNA molecule. This
facilitates easier fragmentation by the hydroxyl radicals. The
inventors found that with the other reagents held constant and the
reaction run at 45.degree. C. for 30 min., the addition of
denaturants (such as formamide, guanidine chloride, ethyl
carbonate, urethane, carbonic acid, and urea) dramatically
increased the sensitivity of RNA to hydrolysis with OP-Cu.
[0123] For example, when the denaturant urea is utilized,
fragmentation increases substantially, as shown by the increase in
fragmentation (FIG. 4B, b, c, e) and the decrease in hybridization
signal (Table 6, treatments 1, 2 and 4). Specifically, when 3.5M
urea is included in the reaction and the hydrogen peroxide
concentration is lowered 10-fold to 10 mM, the same degree of RNA
fragmentation is obtained (FIG. 4B, d) as well as a higher
hybridization signal (Table 6, treatment 3), compared with the
treatment without urea (FIG. 4B, b and Table 6 treatment 1). For
the OP-Cu reaction run at 45.degree. C. for 30 min., 3.5M urea with
10 mM H.sub.2O.sub.2 gave the highest hybridization signal (Table
6, treatment 3). When the OP-Cu reaction is run at 45.degree. C.,
the optimal condition is a 3.5M concentration of urea.
[0124] When the OP-Cu reaction (FIG. 7) was run at 95.degree. C.,
urea is not necessary. FIG. 7A is a fluorescence image while FIG.
7B is a ethidium bromide stain gel image for the OP-Cu and Fe-EDTA
reactions. Lanes a and c show the reaction occurring in the absence
of urea and lanes b and d depict the reaction with 3.5 M urea
present. For the Fe-EDTA protocol run at 95.degree. C. for 10 min.,
urea resulted in an inhibition of fragmentation (FIG. 7d).
Therefore for the Fe-EDTA reaction run at 95.degree. C. urea was
not necessary.
Reduction Step
[0125] An experiment was run for both the OP-Cu and the Fe-EDTA
protocols in which the reductant, NaCNBH.sub.3, was included in the
reaction with all of the other reagents. For the OP-Cu protocol,
the inclusion of the reductant in the reaction step produced a
higher level of fragmentation (FIG. 6A, b) to the standard protocol
(FIG. 6A, a) as well as a 1.5 fold increase in hybridization
signal. This change in the standard OP-Cu protocol reduced the time
required for the protocol by 30 minutes.
[0126] For the Fe-EDTA protocol, the inclusion of NaCNBH.sub.3 in
the reaction step produced an equivalent level (FIG. 6A, d) of
fragmentation to the standard protocol (FIG. 6A, c). Also, the
inclusion of the reductant resulted in a decrease in the
hybridization signal 3-4 fold. At the same time, complete exclusion
of the reduction step from the Fe-EDTA protocol resulted in no
changes in labeling (FIG. 6B) or in fragmentation (FIG. 6C) of
RNA.
[0127] While the invention has been described with reference to
details of the illustrated embodiment, these details are not
intended to limit the scope of the invention as defined in the
appended claims.
* * * * *